Snow Is Coming - What’s That Have to Do With Microbes?

Jan. 11, 2019

In 1978, wheat fields in Montana were supposedly free of Pseudomonas syringae, the bacterium that causes bacterial leaf blight. The field was planted with seeds free of P. syringae. The soil also didn’t contain the pathogen, and irrigation water was also not a source. But these farmlands were still getting infected.

The Discovery of Pseudomonas syringae in the Air

David Sands, a plant pathology professor at Montana State University, had a suspicion: the source of infection had to have come from the air.

He got on a plane, flew above the croplands, opened a window, and held petri dishes outside to expose them to air.

Sure enough, what grew on the dishes was Pseudomonas syringae.

P. syringae wreaks havoc in many ways. It can invade plants through wounds, produce toxins to suppress host defenses, and can cause frost damage in plants (Figure 1). The ice damages the plant’s cell walls allowing the bacterium to gain access to the interior.

Figure 1. Pseudomonas syringae is responsible for frost on plants
Figure 1. Pseudomonas syringae is responsible for frost on plants

Sands knew that P. syringae acts as an ice nuclei on plant surfaces – it helps water molecules condense and eventually form ice. This had Sands thinking: what if the bacterium being kicked up into the atmosphere played a role in the bio-precipitation cycle by helping water molecules condense into clouds? However, Sand’s hypothesis garnered little attention.

Biological ice nuclei and their contribution to snowfall…

Over 20 years later, Sands had a conversation with Brent Christner, an assistant research professor at Montana State University at the time, about the bacteria he found in the sky. This conversation sparked a new idea about bacteria serving as ice nucleators and the team wanted to answer the Sand’s hypothesis from 20 years ago: do bacteria help form clouds?

While the freezing point of water is 0˚C, pure water can actually persist without freezing down to -37˚C. In order to freeze above -37˚C, the water needs a nucleus around which it can assemble in a crystal structure to create a solid. Dust, soot, and other inorganic matter have been shown to act as ice nucleators.

But role of biological ice nuclei in weather was unclear. What they did know was that on plant surfaces, P. syringae can catalyze ice formation at temperatures as high as -2˚C.

To see whether bacteria can act as cloud condensation nuclei the team skied around the world to collect snow and ice: France, Montana, the Yukon, even Antarctica. Then, they counted inorganic particles and cells by flow cytometry. Because ice nucleation activity requires an intact cell, the team treated the cell samples with lysozyme to disrupt the cell membrane. If the lysozyme treatment inhibits the ice nucleation activity, then they could show that cells could serve as ice nucleation sites. The team also hypothesized that ice nucleators of biological origins could be heat inactivated while mineral ice nucleators could not. After treating the samples with heat, they found that it inactivated 69-100% of ice nucleating activity.

Of the 19 fresh snowfalls they analyzed, they found that biological ice nucleators were everywhere. The samples they collected came from seasons and locations devoid of deciduous plants indicating that the ice nucleators traveled long distances and maintained their activity in the atmosphere.

Biological ice nucleators were first described nearly 40 years ago, but it wasn’t until this ski trip in 2008 that there was an estimate for the biological contribution in precipitation.

… And Hailstorms

While that particular study did not specifically mention what species of microbes were found in the snow, another one did. Sands and other colleagues from MSU and Ohio State turned to hailstones collected from three northern Rocky Mountain storms in Montana.

Through a carefully orchestrated process using hot wires, scraping, and ethanol washes, the team was able to separate the various layers of the hailstone from the “embryo,” the frozen raindrop around which the hailstone forms.

The outer layers of the hailstone contained relatively few bacteria, but within the embryo, things got more complex. The team plated the embryo meltwater on three types of media: R2A media (efficient at culturing stressed organisms), nutrient agar (rich medium), and King’s B Medium (selective media for P. syringae) and identified isolates using 16S rRNA sequencing. What they found was a wealth of bacteria within the embryos – each of the embryos contained 12-535 colony forming units of bacteria. Testing a subset of the isolated bacteria, they found that one was an ice nucleator related to Pantoea stewartii, a known ice nucleator.

The researchers only tested 2% of all CFUs obtained during culturing, so it’s therefore possible that there were many more ice nucleators in the hailstone samples. But besides P. stewartii, they found 21 other species of bacteria in just 2% of the samples.

How Bacteria Form Ice

So what’s behind the ice forming phenomenon? When Sands began his investigation into the role of bacteria in the precipitation cycle, researchers were already beginning to identify the source of biological ice nucleation. A team of researchers from the University of Wyoming had already been studying P. syringae and its role in ice nucleation. This group led by Daniel Caldwell found that broth cultures of P. syringae isolated from decaying leaves freeze at warm temperatures (-1.8 to 3.8˚C). The freezing only happened when the cells were intact as chemical treatment and physical destruction of the cells destroyed this activity. Yet, the identity of the catalyst was unknown.

In 1986, the protein behind ice nucleation was revealed: InaZ (Figure 2). But it wasn’t until 2016 that scientists found out how P. syringae actually forms ice by zooming in to the bacterium-liquid interface. Using an imaging technique called interface-specific sum frequency generation (SFG) spectroscopy, the team was able to show that InaZ can change the position of water molecules nearby so that they would fit into a lattice pattern like they do in ice. What’s more, InaZ actually removes heat from the surround water molecules, further helping ice formation.

Figure 2. Side and top view of the InaZ dimer.
Figure 2. Side and top view of the InaZ dimer.

At this point, you may be wondering whether we can use P. syringae to make snow or control the weather. Indeed, P. syringae-derived protein additives in artificial snow help snow machines keep the slopes snowy at higher temperatures. Current methodologies for cloud seeding typically use silver iodide, potassium iodide, and dry ice as nuclei. China cleared the skies before the Beijing Olympics by making it rain early, and the luxury holiday company Oliver’s Travels offered in 2015 a guarantee of “fair weather and blue skies” for £100,000. Cloud seeding can also be used to provide rain to areas of drought.

But adding P. syringae to the mix? Proceed with caution. Sands thinks that too much ice-nucleating bacteria can “constipate the clouds.” In other words, if there are too many ice nuclei, none of them grow large enough to make it rain.

By coaxing liquid into ice, biological ice nucleators greatly influence climate and precipitation. The next time you’re building a snowman, having an epic snowball fight, or skiing through some artificial snow, you can thank these biological ice nucleators for making your winter wonderland possible.

Further reading:

The association between bacteria and rain and possible resultant meteorological implications. Idojaras, 1982.

Biological ice nucleation initiates hailstone formation. Journal of Geophysical Research: Atmosphere, 2014.

Ice-nucleating bacteria control the order and dynamics of interfacial water. Science Advances, 2016.

Ubiquity of Biological Ice Nucleators in Snowfall. Science, 2008.


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Author: Jennifer Tsang, Ph.D.

Jennifer Tsang, Ph.D.
Dr. Jennifer Tsang works in science communications and marketing and writes her own microbiology blog called "The Microbial Menagerie." She completed a Ph.D. in microbiology studying bacterial motility.